The Aryl Hydrocarbon Receptor. The aryl hydrocarbon receptor (AhR) is a ligand inducible transcription factor mediating a broad spectrum of biological processes upon binding to its ligand. Besides induction of enzymes in the cytochrome P450 family, the receptor appears involved with cell proliferation, tumor promotion, immune suppression, vitamin A depletion, and developmental and reproductive abnormalities (Fletcher, et al., Toxicol. Sci. 62(1):166-175, 2001; Safe, Toxicol. Let. 120:1-7, 2001; Gu, et al., Ann. Rev. Pharmacol. Toxicol. 40:519-561, 2000; Poellinger, Food Add. Contam. 17(4):261-266, 2000; Schmidt and Bradfield, Ann. Rev. Cell Dev. Biol. 12:55-89, 1996; Whitlock, et al., Drug Metabol. Rev. 29:1107-1127, 1997). The liganded receptor also causes cell cycle arrest, apoptosis, adipose differentiation, and anti-estrogen effects (Bonnesen, et al., Cancer Res. 61(16):6120-6130, 2001; Elferink, et al., Mol. Pharmacol. 59(4):664-673, 2001; Shimba, et al., J. Cell Sci. 114(15):2809-2817, 2001; Shimba, et al., Biochem. Biophy. Res. Com. 249(1):131-137, 1998; Safe, supra, 2001; McDougal, et al., Cancer Res. 61(10):3902-3907, 2001; McDougal, et al., Cancer Lett. 151:169-179 2000; Elizondo, et al., Mol. Pharmacol. 57(5):1056-1063, 2000; Puga, et al., J. Biol. Chem. 275(4):2943-2950, 2000; Alexander, et al., J. Cell Sci. 111(Part 22):3311-3322, 1998). The presence of the receptor was proposed and evidenced in 1970's (Poland, et al., J. Biol. Chem. 251:4936-4946, 1976). The coding sequence for the receptor was cloned in 1990's and revealed that the AhR is a member of an emerging basic Helix-Loop-Helix/Pas-Arnt-Sim (bHLH/PAS) transcription factor super family (Burbach, et al., Proc. Natl. Acad. Sci. USA 89:8185-8189, 1992).
The bHLH/PAS Super Family of Transcription Factors. The bHLH/PAS super family includes Drosophila Per, Arnt (Ah receptor nuclear translocator, the dimerization partner of AhR and others), SIM1, SIM2, TRH, ARNT-2, the hypoxia inducible factor-1 (HIF-1α), SRC-1, TIF2, RAC3, MOPs 2-5 (Gu, et al., supra, 2000; Hogenesch, et al., J. Biol. Chem. 272:8581-8593, 1997; Wilk, et al., Genes Dev. 10:93-102, 1996), and endothelial PAS domain protein (EPAS-1) (Tian, et al., Genes Dev. 11:72-82, 1997). These bHLH proteins contain the 300 amino acid PAS domain, composed of two 50 amino acid degenerate direct repeats (Burbach, et al., supra, 1992; Dolwick, et al., Mol. Pharmacol. 44:911-917, 1993; Dolwick, et al., Proc. Natl. Acad. Sci. USA 90:8566-70, 1993). The basic region is important for DNA binding, and the HLH and PAS domains are involved in dimerization, and for AhR, in ligand binding (Swanson and Bradfield, Pharmacogenetics 3:213-230, 1993). The transactivation domains of the AhR and ARNT map to their carboxyl termini (Jain, et al., J. Biol. Chem. 269:31518-31524, 1994). Members of this super family are master developmental regulators and it is intriguing to speculate similar roles for AhR and ARNT. Besides with AhR, ARNT forms heterodimers also with HIF-1α, PER, SIM, MOP2 (Hogenesch, et al., supra, 1997), and EPAS-1 (Tian, et al., supra, 1997) and an ARNT-related protein is postulated to heterodimerize with TRH (Wilk, et al., supra, 1996). This promiscuity of ARNT indicates AhR-independent roles for ARNT and suggests the possibility of cross talk between AhR and the other bHLH/PAS signaling pathways.
The Homeostatic Response to Hypoxia: Role of HIF-1a/ARNT-Mediated Gene Expression. Vertebrates require molecular oxygen for vital metabolic processes. Homeostatic responses elicited by hypoxia include erythropoiesis, angiogenesis, and glycolysis. These adaptive responses serve to increase oxygen delivery or activate alternative metabolic pathways that do not require oxygen in hypoxic tissues. In response to hypoxia, HIF-1α translocate into the nucleus where they form heterodimers with ARNT (Gradin, et al., Mol. Cell. Biol. 16(10):5221-31, 1996; Schmidt and Bradfield, supra, 1996). The HIF-1α/ARNT heterodimers bind to hypoxia response elements increasing transcription of genes involved in maintaining oxygenation of tissues. The hypoxia-inducible gene products include erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glycolytic enzymes (Maltepe and Simon, J. Mol. Med. 76(6):391-401, 1998).
The Mode of Action of AhR/ARNT Signaling Pathway. The cytoplasmic form of AhR is associated with 2 molecules of heat shock protein (hsp90) and some other cellular factors (Poellinger, supra, 2000; Whitlock, Ann. Rev. Pharmacol. Toxicol. 30:251-277, 1990). After ligand binding, the hsp90 and the other factors dissociate and AhR is activated. The activated AhR then translocates into the nucleus and dimerizes with its partner ARNT (Probst, et al., Mol. Pharmacol. 44:511-518, 1993). AhR/ARNT heterodimers recognize and bind the so-called xenobiotic response elements (XREs) found in promoters of AhR controlled genes to alter gene expression (Whitlock, supra, 1990). Another potential mechanism involves competition between AhR and either HIF-1α and/or EPAS-1 for dimerization with ARNT. Since AhR, HIF-1α and EPAS-1 require dimerization with ARNT to control the expressions of their target genes, activation of AhR might reduce the availability of free ARNT to such an extent that it becomes rate limiting for other signaling pathways. Decreased availability of ARNT could lead to decreased expression of vital hypoxia-regulated genes and angiogenesis blockage, for example, by inhibiting HIF-1α signaling (Gradin, et al., supra, 1996; Schmidt and Bradfield, supra, 1996).
The Known AhR Ligands. Among the first discovered human-made ligands for the AhR are the chemicals known as polycyclic aromatic hydrocarbons such as 3-methylcholanthrene and benzo[α]pyrene. A much more potent and higher affinity ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was discovered (Poland and Glover, Mol. Pharmacol. 9:736-747, 1973). Another structural group of compounds, halogenated aromatic hydrocarbons, was recognized as the receptor ligands. The compounds with different structural characteristics from the groups mentioned were also found to have binding affinity to AhR. This group is represented by bilirubin (Phelan, et al., Arc. Biochem. Biophy. 357(1):155-163, 1998; Sinal and Bend, Mol. Pharmacol. 52(4):590-599, 1997), lipoxin A(4) (Schaldach, et al., Biochem. 38(23):7594-7600, 1999), brevetoxin-6 (Washburn, et al., Arc. Biochem. Biophy. 343(2):149-156, 1997), diaminotoluene (Cheung, et al., Toxicol. Appl. Pharmacol. 139(1):203-211, 1996), and YH439, a thiazolium compound (Lee, et al., Mol. Pharmacol. 49(6):980-988, 1996). Among most of the human-made AhR ligands, TCDD is one of the most potent agents for AhR and is the prototype compound used to study the mechanism of AhR action and dioxin toxicity. The term “dioxins” has been used to refer to any of the PCDDs (polychlorinated dibenzo-p-dioxins), PCDFs (polychlorinated dibenzofurans), or PCBs (polychlorinated biphenyls) that cause the same biological responses, by the same mechanism as TCDD.
The AhR Ligands with an Indole Moiety. The other recognized AhR ligands with an indole moiety are of special interest. This group consists of tryptamine, indole acetic acid (Heathpagliuso, et al., Biochem. 37(33):11508-11515, 1998), indole-3-carbinol and its derivatives (Stephensen, et al., Nutr Cancer Internatl. J. 36(1):112-121, 2000; Chen, et al., Biochem. Pharmacol. 51(8):1069-1076, 1996; Vasiliou, et al., Biochem. Pharmacol. 50(11):1885-1891, 1995; Liu, et al., Carcinogenesis. 15(10):2347-2352, 1994; Jellinck, et al., Biochem. Pharmacol. 45(5):1129-1136, 1993), and indolo[3,2-b]carbazole (ICZ) (Chen, et al., J. Biol. Chem. 270(38):22548-22555, 1995; Kleman, et al., J. Biol. Chem., 269(7):5137-5144, 1994). Closely related to ICZ, 6-formylindolo[3,2-b]carbazole derived from tryptophan by UV oxidation has higher affinity than that of TCDD to the receptor (Rannug, et al., Chem. Biol. 2(12):841-845, 1995; Rannug, et al., J. Biol. Chem. 262:15422-15427, 1987). Some of the indole derived AhR ligands displayed their interesting properties: binding to the receptor, low toxicity, antiestrogenic and antitumorigenic. Actually, clinical studies have been launched for indole-3-carbinol as an anticarcinogenic and antitumorigenic remedy for patients with high risk of tumorigenesis (Preobrazhenskaya and Korolev, Bioorganicheskaya Khimiya. 26(2):97-111, 2000).
Identity of the Endogenous AhR Ligand and Physiological Functions of the Ah Receptor System Are not Resolved. Okamoto, et al. (Okamoto, et al., Biochem. Biophys. Res. Commun. 197:878-885, 1993) observed that exposure of adult male rats to hyperoxia (95% oxygen) caused induction of CYP1A1 in the lung and CYP1A1 and 1A2 in the liver. The induction of CYP1A1/1A2 is usually associated with the binding of AhR to its ligands. One hypothesis to explain CYP1A1/1A2 induction by hyperoxia is that an endogenous ligand(s) of the AhR is produced by hyperoxia, which activates the transcription of CYP1A1/1A2 genes (Okamoto, et al., supra, 1993). Recently two human urinary products were isolated that bind to the AhR (Adachi, et al., J. Biol. Chem. 276(34):31475-31478, 2001). Whether those products are endogenous ligands or not is undetermined because the identified compounds are indigo, a commonly used fabric dye, and indirubin, an isomer of indigo. Since they were isolated from urine, the question of whether they are urinary excretion products remains unanswered. Similarly, the bilirubin-related compounds (Phelan, et al., supra, 1998; Sinal and Bend, supra, 1997) and lipoxin A(4) (Schaldach, et al., supra, 1999) are certainly endogenous in nature but whether they are the true ligands for the AhR has not yet been resolved. The response and affinity for the AhR appear to be, in fact, quite low for these compounds.
The generation of AhR-deficient mice illustrates possible physiological functions of the receptor in liver, heart, ovary, and the immune system, even though it is not conclusive at this point (Benedict, et al., Toxicol. Sci. 56(2):382-388, 2000; Poellinger, supra, 2000; Mimura, et al., Gene. Cell. 2:645-654, 1997; Schmidt, et al., Proc. Nat. Acad. Sci. USA 93:6731-6736, 1996; Fernandez-Salguero, et al., Science 268:722-726, 1995). The significance of those findings is that they demonstrate a need for a functioning AhR signaling pathway in animal physiology. It is probable that endogenous AhR ligands in animal tissues are involved in carrying out this AhR signaling function.
Importance of Identifying the Endogenous AhR Ligands. Studies with human-made AhR ligands on this receptor system greatly advanced our understanding in this system. It is clear, however, that the AhR did not develop in an evolutionary sense to react to manufactured chemical agents. It is reasonable to suspect that there must be an endogenous ligand for the AhR, which should be nontoxic at tissue concentrations normally encountered in the body, rapidly cleared by metabolism, and utilized to activate the AhR only transiently in a regulatory capacity. Also, evidence shows that the different outcomes of the ligand-receptor mediated signaling processes are possible and dependent upon the nature of the ligands. A decisive factor dictating the consequences in the ligand-receptor mediated signal transducing systems is the final three dimensional conformation of the liganded receptor assumes because that conformation determines the ways the liganded receptor interacts with numerous other factors to transduce signals. Given the amino acid sequence of the receptor, the final three-dimensional structure of the liganded receptor is solely dependent on the structure of the ligand, which ultimately dictates the biological outcomes of the signaling system. To completely understand the physiological functions of the Ah receptor system and the potential therapeutic benefits this system may offer, the identification and synthesis of the AhR ligand is an absolute necessity.